Cullin-ring based ubiquitin ligases (CRLs) constitute one of the largest classes of E3 ubiquitin ligases in mammalian cells, yet many aspects of the mechanisms by which these E3s are regulates and how they their substrates ultimately are degraded by the proteasome remain unknown. In the previous funding cycle, we have explored the functions of Cul3-BTB protein and Cul4-Ddb1DCAF E3s through structural, mechanistic, and genetic approaches. In this renewal, we seek to examine two emerging themes in the ubiquitination field. Theme 1 concerns the question of how one goes about defining the entire repertoire of genes that are require to signal ubiquitination of a substrate, perform the ubiquitination event, and then target the protein to the proteasome. This question is being analyzed in the context of the replication licensing protein Cdt1 and its DNA damage-dependent turnover via the Cul4-Ddb1Cdt2 E3. We have recently completed a genome wide screen for genes whose depletion by RNAi blocks UV-dependent Cdt1 turnover. This screen has revealed 600 candidate genes currently being validated, including near saturation of the known genes involved directly in Cdt1 ubiquitination and proteolysis. A number of known and candidate DNA damage genes were identified, allowing us to place these genes in a hypothetic pathway upstream and downstream of Cdt1 ubiquitination. Through a series of secondary assays, Aim 1 will order validated genes into a signaling pathway, and experiments in mammalian cells and in Xenopus egg extracts will define the mechanisms involved for a subset of genes acting at distinct points in the pathway, including components that may link ubiquitinated Cdt1 to the proteasome. Theme 2 addresses the general question of how ubiquitinated substrates are targeted to the proteasome. There is accumulating evidence in the field indicating that "facilitator" proteins, including proteins with various sorts of ubiquitin binding domains, promote the recognition of ubiquitinated proteins by the proteasome. We have adapted and further developed emerging technologies which allow us to measure the relative rates of turnover of individual substrate in the presence and absence of RNAi targeting components of the degradation machinery. Using this technology in the context of a panel of substrates of the SCF2-TRCP complex, Aim 2 will perform a combinatorial genetic analysis of candidate "facilitator" proteins in order to uncover relevant and potentially redundant "facilitator" proteins for particular substrates, thereby revealing the underlying specificity in substrate recruitment to the proteasome. Using the Global Protein Stability (GPS) system, we will identify targets of the proteasome-associated Rpn10 "facilitator" protein from a library of cells expressing 12,000 human genes, thereby defining the repertoire of ubiquitination targets that require Rpn10 for their turnover. In vitro reconstitution experiments will attempt to recapitulate in vivo specificity of facilitators in an in vitro setting. PUBLIC HEALTH RELEVANCE: Modification of proteins by ubiquitin constitutes a primary mode of protein regulation in cells and underlies diverse signaling pathways. Many human diseases reflect disruption in the pathways that control turnover of cellular proteins. This proposal seeks to understand in greater detail the pathways that control protein ubiquitination and how substrates are targeted to the proteasome.